EA035485B1 - Gene electrotransfer into skin cells - Google Patents

Abstract

The present invention relates to a method for preventing, suppressing or reducing tumor in a subject in vivo and to a method of antitumor vaccination or gene therapy of a subject in vivo, that provide administering a nucleic acid encoding a tumor antigen and inducing a cellular immune response. For better efficiency, the nucleic acid is injected by intradermal injection and electrical permeabilization of skin cells with a single pulse of high voltage field strength of 1100 to 1400 V/cm and of duration of 10 to 1000 s; followed by a single pulse of low voltage field strength of 100 to 200 V/cm and of duration of between 300 and 800 ms.

Description

The present invention relates to the field of electropermeabilization of cells for gene transfer.

Background Art

Electropermeabilization of cells, i.e. permeabilization of cells by local delivery of electrical impulses (EP) is increasingly used to treat and prevent a variety of pathologies in humans and animals, including cancer.

The cell membrane defines the boundaries of two compartments, the cytoplasm and the extracellular environment, which contain different concentrations of ions, thereby creating a transmembrane potential difference. Application of an electric field to cells induces a transmembrane potential, which is superimposed on the resting potential (Mir et al., 2005). Above the threshold value, a temporary permeabilization occurs, which leads to the exchange of molecules between the cytoplasm and the external environment. This phenomenon, which occurs after the application of an electrical impulse (EP) to cells and leads to a loss of membrane permeability, is called electropermeabilization. This technology has been used for three decades to enhance the absorption of molecules that are not otherwise absorbed by cells.

Although the specific mechanism of electropermeabilization is still under discussion, this technology has indicated the path for many biomedical applications, in particular for cancer therapies (Breton & Mir, 2011). One of the therapies, called antitumor electrochemotherapy, consists of combining an electrical pulse (EP) applied directly to the tumor site with the administration of bleomycin or cisplatin, which do not diffuse spontaneously (or weakly diffuse) across the plasma membrane (Mir et al., 1991; Mir , 2006). Upon entry into electrically permeabilized cells, these two drugs generate DNA damage and trigger cell death. Not only drugs, but also nucleic acids, which are non-penetrating molecules (Satkauskas et al., 2002; Andre & Mir, 2010), can be electrotransferred into cells using electropermeabilization. DNA has been successfully transferred into various tissues of living animals, including skin, muscle, liver, tumor, cornea, lungs, kidneys, brain, bladder and testis (reviewed in Andre et al., 2008; Gothelf & Gehl, 2010 ). One of the promising applications of the gene electrotransfer method relates to the field of DNA vaccination. Indeed, DNA vaccination has generated a lot of interest since the early 90s. For the first time, Wolff and his co-workers were able to transfer DNA into the muscles of an animal. Once transfected, the DNA molecules have imparted the ability of target cells to produce the encoded protein (Wolff et al., 1990). Tang et al. showed that a protein encoded by DNA transferred into skin cells by a biolistic method was able to trigger an immune response (Tang et al., 1992), and Barry et al. it has been shown that gene vaccination with a plasmid encoding a pathogen protein protected animals against infection by the corresponding pathogen (Barry et al., 1995). This technology has been used in a variety of applications ranging from laboratory instruments to licensed veterinary vaccines (Anderson et al., 1996), and is currently in development for the treatment of various acquired pathologies such as cancer, malaria, hepatitis B and C. , or preventing certain viral infections, such as influenza or human immunodeficiency virus (clinicaltrials.gov) (Bergman et al., 2003).

Thus, DNA vaccines have advantages in terms of manufacture, availability and cost-effectiveness over other vaccine manufacturing technologies (Liu, 2011). Despite the fact that DNA vaccines provide an accurate and flexible strategy for delivering antigens to immune cells and elicit a specific immune response, initially there were difficulties in transferring this technology from small rodents to larger animals and ultimately to patients. (Rochard et al., 2011). In fact, DNA vaccines have been found to be weakly immunogenic, in part due to the low uptake of DNA molecules by cells at the site of vaccine administration. This problem was solved by the use of gene electrotransfer, which significantly improved the characteristics of DNA vaccines (Li et al., 2012; Gothelf & Gehl, 2012).

However, the use of electrotransportation as envisioned in vaccination strategies requires a very specific procedure. Electrotransport is a multistep process based on two types of electrical impulses (EPs) (Andre & Mir, 2010; Satkauskas et al., 2005; Favard et al., 2007). First, the DNA is brought into close proximity to the environment of the target cells by injection into the intended site of vaccine administration (skin, muscles), then one or more short (about 100 μs) and intense (about 1000 V / cm) pulses, called high voltage pulses ( HV), reversibly permeabilize the cell membrane. After a certain time interval, one or more long (about a few hundred milliseconds) and less intense (about a hundred volts per centimeter) pulses are applied, called low voltage (LV) pulses. LV pulses are designed for electrophoretic movement of DNA through the extracellular matrix up to contact with the electrophoretic membrane. So far, there is no consensus on how DNA molecules cross the plasma membrane and reach the nucleus in order to be perceived by the cell translation mechanism (Escoffre et al., 2009).

Interestingly, after the introduction of DNA followed by electrotransfer, neither animals nor humans were found to have serious side effects (Fioretti et al., 2013). Research has shown that

- 1 035485 after administration, the DNA vaccine was mainly localized around the injection site, its local detection rates dropped rapidly over time, it was not internalized by gonadal tissue (very low risk of germline transfer) and the likelihood of integration was very low because it was not a viral protein was used (Dolter et al., 2011). Moreover, when DNA molecules are used for vaccination in combination with gene electrotransfer, there are no immunity-related problems before or after treatment, unlike viral vectors such as adenoviral vectors, thereby allowing multiple administrations (homologous prime-boost vaccination DNA / DNA or DNA / vector or DNA / protein heterologous prime boost vaccination) (Villemejane & Mir, 2009). Thus, DNA vaccination combined with electrotransfer has attracted interest in the past few years.

In terms of cancer, DNA vaccines are designed to trigger an immune response against tumor-specific or tumor-associated antigens (Stevenson & Palucka, 2010). Indeed, cancer cells trick the immune system, which may not always effectively initiate an immune response due to many complex mechanisms, such as self-tolerance (Bei & Scardino, 2010), various immunosuppression mechanisms, including regulatory T cells or suppressor dendritic cells of myeloid origin (moDC ) (Lindau et al., 2013), molecules such as CTLA-4 expressed on the surface of immune cells (Kolar et al., 2009; Shevach, 2009), and PD-1 / PD-1L interactions (Keir et al. , 2008).

The ultimate goal of an effective DNA vaccine delivered by electrotransfer technology should be to generate the correct type of immune response against the antigen encoded by the plasmid of interest. Although electrotransport technology has been described with sufficient detail for the intramuscular route of administration (patent application WO 2007/026236) (Mir et al., 2005; Andre & Mir, 2010), little information is available on the parameters of electrotransportation into the skin for vaccination purposes. The immune response must be sufficiently intense and prolonged to generate positive therapeutic effects in patients with a particular pathology. It should be noted that the intensity of the immune response depends, at least in part, on the level of antigen expression (Lee et al., 1997; Kirman & Seder, 2003), which itself is closely related to the efficiency of gene transfer. With regard to the efficiency of gene electrotransfer into the skin, it depends on several parameters, including the intensity of the electrical impulses (EP) and the type of electrodes used to deliver them (Gothelf & Gehl, 2010).

Brief description of the invention

The inventors have found that the efficiency of electrotransfer to the skin can be improved by using a specific combination of high and low voltage pulses.

The present invention relates to a method for preventing, suppressing or reducing a tumor in a subject in vivo by introducing a nucleic acid encoding a tumor antigen and inducing a cellular immune response. In this method, nucleic acid is administered by intradermal injection and electrical permeabilization of skin cells using a single pulse of high voltage electric field in the range from 1100 to 1400 V / cm and duration from 10 to 1000 μs;

the subsequent single pulse of a low voltage electric field in the range from 100 to 200 V / cm and duration from 300 to 800 ms.

In one embodiment of the method, non-invasive plate electrodes are used.

An embodiment of the method is also provided where the high voltage pulse and the low voltage pulse are separated from each other by a time interval. The duration of this time interval can be from 300 to 3000 ms.

Another object of the present invention is a method for antitumor vaccination or gene therapy of a subject, comprising administering a nucleic acid encoding a tumor antigen and inducing a cellular immune response. This method provides for the introduction of nucleic acid by intradermal injection and electrical permeabilization of skin cells using a single pulse of high voltage electric field in the range from 1100 to 1400 V / cm and duration from 10 to 1000 μs;

the subsequent single pulse of a low voltage electric field in the range from 100 to 200 V / cm and duration from 300 to 800 ms.

In one embodiment, the high voltage pulse and the low voltage pulse are separated by a time interval.

The cellular response elicited by the methods of the invention may be a CD8 T cell response.

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Brief Description of Drawings

FIG. 1 is a schematic representation of the INVAC-1 plasmid DNA vector. Bases 1-3478: vector NTC8685-eRNA41H-HindIII-XbaI (NTC); bases 3479-3484: HindIII cloning site (NTC / Invectys); bases 3485-6967: transgene Ubi telomerase (Invectys); bases 6968-6973: XbaI cloning site (Invectys / NTC); bases 6974-7120: vector NTC8685-eRNA41H-HindIII-XbaI (NTC).

FIG. 2A and B are graphs that show that electrotransfer is preferred for gene transfer and immunization. (A) Image of bioluminescence intensities in C57BL / 6J mice after injection of pCMV-luc with or without subsequent electrical impulse (EP) or without subsequent electrical impulse (EP), n = 5 mice for pCMV-luc injection ID only, n = 10 (from 5 mice, two treatments per mouse) for ID injection pCMV-luc + EP. (B) Frequency of hTERT-specific INFy + CD8 T cells found in C57BL / 6J mice vaccinated with INVAC-1 with or without subsequent EP, n = 6-8 mice. The columns represent averages. * p <0.05, ** p <0.01, Wilcoxon-Mann-Whitney test.

FIG. 3A and B are graphs that show the selection of the best electrodes. (A) Image of bioluminescence intensities in C57BL / 6J mice after electrotransfer of pCMV-luc using three types of electrodes, n = 14 mice for ID pCMV-luc injection only, n = 8-10 (4 to 5 mice, two treatments per mouse ) for ID in HLA-B7 mice vaccinated with INVAC-1 using three types of electrodes, n = 3 mice for PBS control immunization, and n = 4-9 mice for INVAC-1-mediated immunization. The columns represent averages. * p <0.05, *** p <0.001, Kruskal-Wallis test with Dunn's test for multiple comparisons.

FIG. 4 is a set of images showing the localization of luciferase gene expression in C57BL / 6J mice after ID injection and pCMV-luc electrotransfer using plate electrodes.

FIG. 5 is a graph that shows the determination of the optimal HV pulse intensity in C57BL / 6J mice for electrotransfer of intradermally injected pCMV-luc using plate electrodes, n = 24 mice for pCMV-luc injection ID only, n = 8 (from 4 mice, two treatments per mouse) for ID injection pCMV-luc + EP. The columns represent averages. * p <0.05, ** p <0.01, *** p <0.001, Kruskal-Wallis test with Dunn's test for multiple comparisons.

FIG. 6A and B are graphs that show the selection of the best combination of HV-LV pulses in C57BL / 6J mice. (A) Bioluminescence obtained after ID injection of pCMV-luc at various combinations of HV-LV pulses, n = 30 mice for ID pCMV-luc injection only and n = 6 (from 3 mice, two treatments per mouse) for ID pCMV-luc injection luc + EP. (B) Frequency of hTERT-specific IFNy + CD8 T cells found in C57BL / 6J mice vaccinated with INVAC-1 according to various combinations of HV-LV pulses, n = 8 mice for control immunization with PBS, and n = 5 mice for INVAC-1 -mediated immunization. The columns represent averages. * p <0.05, ** p <0.01, *** p <0.001, Kruskal-Wallis test with Dunn's test for multiple comparisons.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term HV means high voltage and the term LV means low voltage.

As used herein, the term skin refers to the skin of an animal, for example, a human or non-human mammal, such as a rodent (for example, a mouse, rabbit, or rat), dog, cat or primate, horse, goat, pig, sheep, cow, etc. In a preferred embodiment, the nucleic acid is transferred to the cells of the dermis. The skin cells into which the nucleic acid according to the invention has been transferred are preferably dendritic cells, but can also include keratinocytes, melanocytes, fibroblasts, or myeloid or lymphoid cells. The term "nucleic acid" means any nucleic acid of interest, in particular any nucleic acid capable of expressing a protein of interest. The nucleic acid can be single-stranded or double-stranded DNA or RNA (eg, antisense or interfering RNA). Preferably it is DNA, preferably double stranded DNA. In a preferred embodiment, the nucleic acid is a DNA expression vector of a type well known in the art. Typically, the expression vector contains a promoter operably linked to a DNA sequence that encodes a protein of interest.

The term TERT refers to telomerase reverse transcriptase, which is a major determinant of telomerase activity, including wild-type telomerase or variants thereof.

The term “immunogenic” means that the composition or construct to which it refers, when administered, is capable of eliciting an immune response. The immune response in a subject refers to the development of an

- 3 035485 of an anticipated and adaptive immune response to an antigen, including a humoral immune response, a cellular immune response, or both. The humoral immune response refers to an antibody-mediated response. The cellular immune response is a T-lymphocyte-mediated response. It includes the production of cytokines, chemokines, and other similar molecules produced by activated T cells, white blood cells, or both. Immune responses can be determined using standard immunoassays and neutralization assays for detecting humoral immune responses, which are known in the art. With respect to cancer vaccination, the immune response preferably encompasses the stimulation or proliferation of cytotoxic CD8 T cells and / or CD4 T cells and can be determined using immunoassays such as an ELISpot assay, an in vivo cytotoxicity assay, or a cytokine secretion binding assay.

As used herein, the term treatment, or therapy, or immunotherapy refers to any of amelioration, amelioration and / or elimination, reduction and / or stabilization (eg, no progression into later stages) of a symptom, as well as delaying the progression of a disease or symptom. In case the disease is cancer, the term includes achieving an effective antitumor response seen in cancer patients.

As used herein, the term prevention or prevention refers to mitigation, amelioration and / or elimination, reduction and / or stabilization (eg, no progression into later stages) of signs prior to the onset of the disease, i.e. any change or symptom (or set of symptoms) that may indicate the onset of a disease prior to the onset of specific symptoms.

The patient or subject is typically a mammalian subject as defined above, preferably a human of any age, sex or severity of the condition.

Electric transfer parameters.

The nucleic acid is preferably intended to be brought into contact with skin cells prior to application of a single LV pulse, and even more preferably prior to application of a single HV pulse. The time interval between nucleic acid injection and electrical impulses, especially between injection and a single HV pulse, is not important. Typically, the time interval (before the HV pulse is applied) after contacting the pharmaceutical composition with skin cells is from a few seconds to 10 minutes, for example from 30 seconds to 5 minutes. A time interval of 5 to 10 minutes before the HV pulse is applied is also acceptable. A nucleic acid or a pharmaceutical composition containing a nucleic acid is brought into contact with skin cells (ie, dermal cells) by intradermal injection.

In a preferred aspect of the invention, the single high voltage pulse preferably has a field strength of 1100 to 1400 V / cm, preferably 1250 V / cm.

A single high voltage pulse can have a duration of 50 to 150 μs, preferably 100 μs.

In a preferred aspect of the invention, the single low voltage pulse preferably has a field strength of 100 to 200 V / cm, preferably 180 V / cm.

A single low voltage pulse may preferably have a duration of 350 to 600 ms, more preferably 400 ms.

In a preferred embodiment, the single high voltage pulse preferably has a field strength of 1100 to 1400 V / cm, preferably 1250 V / cm and a duration of 50 to 150 μs, preferably 100 μs, and the single low voltage pulse has a field strength of 100 to 200 V / cm, preferably 180 V / cm and a duration of 350 to 600 ms, more preferably 400 ms.

In a particular embodiment, when the subject is a human, a single high voltage pulse may have a field strength of 1250 V / cm and preferably a duration of 100 μs, and a single low voltage pulse may have a field strength of 180 V / cm and preferably a duration of 400 ms.

The low voltage (LV) pulse can have the same polarity, or the opposite polarity of the HV pulse. Preferably, the LV unit pulse is a square pulse. It can also be trapezoidal or periodic. Preferably, the unit HV pulse is a square pulse.

The HV and LV pulses can be separated by a time interval, and this time interval is preferably 300 to 3000 ms, preferably 500 to 1200 ms, typically 1000 ms.

The aim of the invention is a method of electroporation as such, which includes placing electrodes near the cells of the dermis containing interstitial nucleic acid, then electrically permeabilizing the cells of the dermis as follows:

first, using a single high voltage electric field pulse in the range from 1000 to 1500 V / cm and duration from 10 μs to 1000 μs;

- 4 035485 then preferably after a certain time interval by means of a single pulse of a low voltage electric field in the range of 50 to 250 V / cm and a duration of 300 to

800 ms, and as a result of these electrical impulses, nucleic acid is transferred into the cells of the dermis.

A programmable voltage generator can be used.

You can use electrodes, which are invasive needle electrodes (such as N-30-4B, IGEA), usually consisting of two rows of four long needles spaced 4 mm apart, or invasive finger electrodes (such as F- 05-OR, IGEA), usually consisting of two rows of three short needles spaced 4 mm apart, but preferably non-invasive plate electrodes (such as P30-8B, IGEA).

The electrodes are positioned in close proximity to the injection site so that the electric field between the electrodes passes through the injection site or area, and the injected fluid diffuses after injection. Preferably, a conductive gel known to the person skilled in the art is used.

In a particular embodiment, the electrodes may be contained in a device through which both administration and electrical stimulation can be performed.

Genetic constructs, immunogenic compositions.

Preferably, the nucleic acid is a genetic construct comprising a polynucleotide sequence encoding a protein of interest and regulatory sequences (such as a suitable promoter (s), enhancer (s), terminator (s), etc.), allowing expression (for example, transcription and translation) of a protein product in a host cell or host organism.

Genetic constructs according to the invention are generally in a form suitable for transforming the desired host cells or host organism, in a form suitable for insertion into the genomic DNA of the desired host cells, or in a form suitable for independent replication, maintenance and / or inheritance in a given cell or organism. For example, the genetic constructs of the invention may be in the form of a vector, such as, for example, a plasmid, cosmid, YAC, viral vector, or transposon. In particular, the vector can be an expression vector, i. E. a vector that can provide RNA transcription and / or protein expression in vivo, especially in dermal cells.

In a preferred, but not limiting aspect, a genetic construct of the invention comprises i) at least one nucleic acid encoding a protein of interest operably linked to ii) one or more regulatory elements such as a promoter and optionally a suitable terminator, and optionally also iii) one or more additional elements of genetic constructs, such as 3'- or 5'-UTR sequences, leader sequences, selection markers, expression markers / reporter genes, and / or elements that can promote or enhance (efficiency) transformation or integration.

It should be understood that the use in accordance with the invention contemplates the case when two or more nucleic acids capable of expressing different active molecules in vivo are used for the manufacture of a pharmaceutical composition. Nucleic acids are selected so that they are complementary and / or act synergistically to treat disease. In this case, the nucleotide sequences encoding different molecules can be under the control of the same promoter or different promoters. Compositions containing the specified nucleic acid (s) or vector (s) can be obtained. In one embodiment, the compositions are immunogenic. They may contain a carrier or excipients that are suitable for administration to humans or mammals (ie, non-toxic). Such adjuvants include liquid, semi-solid, or solid diluents that serve as pharmaceutical carriers, isotonic agents, stabilizers, or any adjuvant.

Non-coding nucleic acids.

In another embodiment, the nucleic acid does not encode any protein of interest, but inhibits or attenuates the expression of the target gene. For example, the nucleic acid can be antisense RNA or interfering RNA. The nucleic acids can be naturally occurring nucleic acid RNA molecules or nucleic acid analogs such as PLNA. In particular, the nucleic acid can be a small interfering RNA (siRNA), for example, a single-stranded or double-stranded RNA, such as a short single-stranded or double-stranded RNA of about 17 to 29 nucleotides in length, preferably about 19 to 25 nucleotides in length, which target target mRNA. In the case of double-stranded RNA, such siRNA contains a sense RNA strand and a complementary antisense RNA strand that are hybridized with each other.

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Vaccination and / or gene therapy.

In a specific embodiment, the nucleic acid is useful in gene therapy and / or vaccination by expressing a protein of interest. In a preferred embodiment, the protein of interest has an immunostimulatory effect, more preferably a vaccine effect. Preferably, a humoral immune response is obtained.

In a preferred aspect, the nucleic acid comprises nucleic acid sequences capable of expressing in vivo in transfected skin cells one or more therapeutically active molecule (s), preferably the protein or proteins of interest.

This active molecule or protein of interest may act in the skin itself and / or outside the skin at another location within the body, for example, on a tumor localized elsewhere in the body if the expressed molecule is active as an anti-tumor vaccine, or an infection localized where - or within the body, if the expressed molecule is active as an anti-infective vaccine.

An example of a therapeutic molecule of interest is the TERT protein useful as an anti-tumor vaccine. It should be appreciated that there is no limitation on the kind of molecules that can be expressed in accordance with the invention, and therefore, a person skilled in the art will be able to carry out the invention using the molecule of interest, knowing its coding sequence, and routine experiments to select the best construct or expression vector.

In one interesting aspect, the nucleic acid, as a therapeutically active molecule, encodes one or more immunogens (or immunogenic peptides, polypeptides, or proteins, including glycoproteins) that are capable of eliciting an immune response in a host. In one embodiment, the immune response is a protective immune response for the host. In this embodiment, the invention relates to the preparation of an immunogenic composition, or a prophylactic or therapeutic vaccine, which is directed against cancer or against microorganisms, for example viruses or bacteria.

By way of example only, the nucleic acid encodes one or more of the immunogens of HIV, HBV, Epstein-Barr virus, pseudorabies virus, synthesia-forming virus, oncovirus, papillomavirus, etc. The skilled artisan has access to nucleic acids encoding molecules of interest for a selected application, for example, the most effective immunogens or combinations of immunogens for a particular disease.

In another embodiment, the immune response results in the production of antibodies, especially polyclonal antibodies, and these antibodies are recovered from the resulting serum and used in a conventional manner.

In yet another embodiment, the nucleic acid encodes an antigenic peptide or protein that, when administered to a subject, eg, mice, triggers the production of specific antibodies.

Treatment of tumors.

In a specific embodiment, a method for preventing or treating a tumor or unwanted cell proliferation (eg, dysplasia) in a patient is described, the method comprising administering an effective amount of a nucleic acid or immunogenic composition to a patient in need thereof using the electroporation method of the invention. The specified nucleic acid or immunogenic composition is administered in an amount sufficient to induce an immune response in a patient.

The tumor can be any unwanted cell proliferation, in particular a benign tumor or a malignant tumor, in particular cancer.

Cancer can be at any stage of development, including the metastatic stage.

Thus, the nucleic acid preferably expresses one or more active molecules selected so that the pharmaceutical composition is effective in reducing, suppressing or regressing tumor angiogenesis, or reducing or suppressing tumor growth, or inhibiting metastasis. For example, a nucleic acid encoding a tumor antigen (eg, TERT) can be used.

In a specific embodiment, the tumor is solid cancer, sarcoma, or carcinoma. In particular, the tumor can be selected from the group consisting of melanoma, brain tumors such as glioblastoma, neuroblastoma and astrocytoma, and carcinomas of the bladder, breast, cervix, colon, lungs, especially non-small cell lung cancer (NSCLC) , pancreas, prostate, head and neck cancer, or stomach cancer.

In another embodiment, the tumor can be a liquid tumor, for example a hematopoietic tumor, lymphoma or leukemia such as lymphocytic leukemia, myeloid leukemia, lymphoma, including Hodgkin's disease, multiple myeloma, malignant myeloma.

While it should be understood that the amount of material required will depend on the immunogenicity of each individual construct and cannot be predicted a priori, the process of determining the appropriate dose for a given construct is straightforward. In particular, a series of increasing doses, for example starting from about 5-30 μg, or preferably 20-25 μg, up to about 500-1000 μg, is administered to the respective species and the final immune response is observed, for example, by detecting a cellular immune response using an ELISpot. IFNY assay (described in the experimental section), by detecting cytotoxic T lymphocyte (CTL) responses using an in vivo lysis assay determined by standard chromium release assays, or by detecting TH (helper T cells) responses using a cytokine release assay ...

In a preferred embodiment, the vaccination regimens comprise one to three injections, preferably repeated after three or four weeks. In a specific embodiment, the vaccination schedule may consist of one or two injections and at least one subsequent cycle three or four weeks later, consisting of three to five injections. In another embodiment, the first dose consists of one to three injections followed by at least a booster dose administered every year or every two or more years. These are examples only, and any other vaccination regimen is encompassed by the present invention.

The present invention is described in more detail using the following non-limiting experiments.

Examples of

Electro-gene transfer procedure for anti-tumor vaccination.

In the present study, the procedure for electrotransfer of genes into the dermis was optimized for the purpose of vaccination against the telomerase tumor antigen. The first evaluation used the luciferase reporter gene to evaluate the efficiency of gene electrotransfer to the dermis as a function of the parameters used. In a second assessment, these parameters were tested for their efficacy in immunizing mice against telomerase epitopes. Used different types of electrodes, non-invasive or invasive, as well as a number of different applied electric fields.

Two important parameters were evaluated, the first of which was the intensity of luciferase expression at the electrotransport site, and the second was the intensity of the vaccine-specific, interferon γ (III) positive CD8 T cells, which represent the type of immune response expected for cancer vaccines (Vesely et al., 2011). Three main factors of electrotransfer were studied: types of electrodes and the impact of HV and LV pulses.

Materials and methods.

Mouse.

HLA-B7 mice are transgenic mice expressing the class I molecule HLA-B * 0702. They are knockout class I molecules H2D ^b and H2K ^b . They were previously described by Rohrlich et al., 2003 and were obtained by internal breeding at The Pasteur Institute. Female C57BL / 6J mice (6-8 weeks old) were obtained from Janvier (Saint-Berthevin, France) or Harlan (Gannat, France) laboratories.

The animals were kept in special pathogen-free conditions in the laboratory of the Pasteur Institute or the Gustave Roussy Institute. All animal experiments were carried out in strict accordance with the ethical guidelines of the European Committee (Directive 2010/63 / EU), and manipulations with animals were carried out in strict accordance with good animal practice.

Plasmids.

pCMV-luc (PF461, Plasmid Factory, Bielefeld, Germany) is a 6233 bp double-stranded plasmid DNA encoding a firefly luciferase reporter gene placed under the control of the cytomegalovirus (pCMV) promoter.

INVAC-1 is a 7120 bp double-stranded plasmid DNA encoding a modified telomerase protein sequence fused to the ubequitin protein sequence. The encoded telomerase protein is enzymatically inactive, but can still elicit immune responses in vivo against telomerase epitopes. The ubiquitintelomerase insert was cloned into the expression vector NTC8685-ERNA41H-HindIII-XbaI, developed by Nature Technology Corporation (Lincoln, Nebraska). The presence of ubiquitin increases the transfer of the telomerase reverse transcriptase (TERT) protein to the proteasome and increases the presentation pathway for peptides derived from TERT, MHC class I (Rodriguez et al., 1997; Wang et al., 2012). The DNA sequence encoding the TERT protein contains a 47 amino acid deletion in the N-terminal region, which includes the nucleolar localization signal. In addition, three amino acids have been removed within the TERT catalytic site (VDD) to eliminate the enzymatic activity of the protein. Plasmid INVAC-1 was stored at -20 ° C in phosphate buffered saline (PBS) at a concentration of 2 mg / ml until use. FIG. 1 shows a map of the INVAC-1 plasmid.

EP generator and electrodes.

Gene electrotransfer was performed using a Cliniporator® (IGEA, Carpi, Italy) delivering HV pulses and LV pulses. The voltage value was set in accordance with the distance

- 7 035485 between two rows of electrodes. Various types of electrodes were used: (1) invasive needle electrodes (N-30-4B, IGEA), consisting of two rows of four long needles spaced 4 mm apart, (2) invasive finger electrodes (F-05 -OR, IGEA), consisting of two rows of three short needles spaced 4 mm apart, (3) non-invasive plate electrodes (P30-8B, IGEA), consisting of two metal plates 1 mm thick and spaced apart from each other from a friend at a distance of 5 mm.

Gene electrical transfer in vivo.

Mice were anesthetized prior to intradermal (ID) injections using an anesthetic gas mixture of 2% isoflurane / oxygen (Abbot, Suresnes, France) or a mixed solution (intraperitoneal method) of 2% xylazine (Rompun, Bayer Sante, Loos, France) and 8% ketamine (Imalgen 1000, Merial, Lyon, France) in PBS according to the individual animal body weight. Intradermal (ID) injection was performed after shaving into the lower side of the animal (bilateral injections) using insulin specific needles 29 G thick. Each animal of the HLA-B7 or C57BL / 6J mouse line received a single dose of DNA corresponding to 100 μg of the INVAC-1 plasmid ( 50 μg in 25 μl PBS on the side) or 10 μg of pCMV-luc plasmid (5 μg in 25 μl PBS on the side).

Immediately after the ID injection, gene electrotransfer was performed using an HV pulse (100 μs duration) followed by one LV pulse (400 ms duration) 1000 ms later. The electrodes were placed so that they surrounded the blister formed by the injection of the plasmid. Finger or needle electrodes were pressed into the skin to a depth of about 5 mm. For the plate electrodes, a conductive gel (Labo FH, gel de contact bleu, NM Medical, France) was used to improve the contact between the metal plates and the skin.

Bioluminescence imaging in vivo and localization of electrical transport.

Two days after electrotransfer of pCMV-luc, C57BL / 6J mice were injected intraperitoneally with 0.15 mg of beetle luciferin (Promega, Lyon, France) per gram of body weight. 20 min after injection, the animals were anesthetized using an anesthetic gas mixture of 2% isoflurane / oxygen, and a luciferase-driven bioluminescence response was detected using an In Vivo Imaging System IVIS 50 (Xenogen, Waltham, USA). To confirm the electrotransfer to the skin, three mice were sacrificed by cervical dislocation 20 min after the injection of luciferin, and the electropermeabilized skin area was removed from the animals. The intensity of bioluminescence was assessed in the skin flap and in the underlying muscles.

Preparation of splenocytes.

14 days after INVAC-1 ID injection and electrotransfer, mice were sacrificed by cervical dislocation and spleens removed. Under sterile conditions, each spleen was pressed through a 70 μm nylon mesh (cell filter, BD Falcon Franklin Lakes, USA) and washed with RPMI complete culture medium (Roswell Park Memorial Institute, medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 1 % sodium pyruvate, 1% penicillin streptomycin and 0.1% β-mercaptoethanol). All components were obtained from Life technologies SAS (Saint-Aubin, France). Splenocytes were purified on a ficoll density gradient (Lymphocyte Separation Medium, Eurobio, Courtaboeuf, France), washed and counted using a Cellometer® Auto T4 Plus counter (Ozyme, Saint-Quentin-en-Yvelines, France) and adjusted to 2 million cells / ml in complete RPMI medium before use in the IFNy ELISpot assay.

HLA-B7 and H2 restricted peptides.

Human TERT (hTERT) peptides, restricted by the class I molecule HLA-B * 0702, have been previously described (Adotevi et al., 2006; Cortez-Gonzalez et al., 2006). Other peptides were predicted by in-silico prediction of epitopes for binding of murine H2K ^b , H2D ^b MHC class I using four algorithms available on the Internet: Syfpeithi (http://www.syfpeithi.de/), Bimas (http: / /www-bimas.cit.nih.gov), NetMHCpan and SMM (http://tools.immuneepitope.org/main/). All synthetic peptides were obtained lyophilized (> 90% purity) from Proimmune (Oxford, UK). Lyophilized peptides were dissolved in sterile water at 2 mg / ml and stored in 35 μl aliquots at -20 ° C until use. Details of the peptide sequences for B7 or H2 restriction are presented below.

H2-restricted hTERT peptides:

H2D ^b : RPIVNMDYV (p660),

H2K ^b : HAQCPYGVL (p429).

HLA-B7 restriction hTERT peptides:

HLA-B7: RPSLTGARRL (p351),

HLA-B7: RPAEEATSL (p277),

HLA-B7: LPSDFKTIL (p1123).

IFNy ELISpot analysis.

Briefly, polyvinylidene fluoride microplates (IFNy ELISpot assay kit, 10x96 assays, Diaclone, Eurobio) were coated overnight with capture antibody (anti-mouse

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IFNy) and blocked with sterile 2% milk in PBS for 2 hours. ELISpot plates were washed and splenocyte suspensions were plated in triplicate at a concentration of 2x10 ⁵ cells / well. Cells were then stimulated with 5 μg / ml of the respective peptides H2 or B7 or 10 μg / ml phorbol-12-myristate-13 acetate (PMA) -ionomycin or mock stimulated with serum-free culture medium. The plates were incubated at 37 ° C in an atmosphere of 5% CO ₂ . After 19 h, spots were detected using an antibody conjugated with biotin to detect IFNy, then streptavidin alkaline phosphatase and a substrate solution 5-bromo-4-chloro-3'-indolyl phosphate p-toluidine salt / nitro blue tetrazolium (BCIP / NBT) were used ... The spots were counted using an Immunospot ELISpot counter and software (Cellular Technology Limited, Bonn, Germany).

Statistical analysis and data processing.

Prism-5 software was used for data processing, analysis and graphing. For statistical analyzes of bioluminescence and ELISpot, the Mann-Whitney-Wilcoxon test or the Kruskal-Wallis test with Dunn's test was used for multiple comparisons depending on the experiment. Significance was established at p-value <0.05.

Results.

Electrotransfer provided optimal in vivo transgenic expression and induction of antigen-specific CD8 T cells.

Plasmids pCMV-luc or INVAC-1 were injected intradermally into the shaved sides of C57BL / 6J mice with or without subsequent application of EP (1 HV pulse at 1000 V / cm for 100 μs, followed by an LV pulse at 140 V / cm for 400 ms after 1000 ms). No irritation was observed after shaving or during and after the electrotransfer procedure. After gene electrotransfer, two parameters were measured: luciferase expression after electrotransfer ID pCMV-luc and the frequency of IFNY-secreting hTERT-specific CD8 T cells after INVAC-1 electrotransfer ID. The expression of luciferase (Fig.2A) and the frequency of IFNY-secreting hTERT-specific CD8 T cells (Fig.2B) were significantly increased when EP was applied immediately after DNA injection (p <0.01 and p <0.05, respectively) compared to animals who received DNA injection without electrotransfer. Thus, electrotransfer induces significant levels of hTERT-specific CD8 T cell responses after ID vaccination with INVAC-1 and significant levels of luciferase expression after ID injection of pCMV-luc.

Selecting the best electrodes for optimal gene transfer and generation of intense cellular immune responses.

Various types of electrodes can be used for in vivo electrotransfer to the skin (Gothelf & Gehl, 2010). Thus, three different types of electrodes (plate electrodes, finger electrodes, and needle electrodes described in the Materials and Methods section) were tested to determine which one is best suited for efficient gene transfer and generation of intense specific cellular immune responses in mice. The results showed that the three types of electrodes significantly enhanced electrotransfer of plasmid pCMV-luc in C57BL / 6J mice compared to animals that received plasmid without EP (Fig. 3A). However, a higher homogeneity of the response was observed for the group of mice subjected to electrotransfer using plate electrodes (p <0.001). Animals electrotransferred using needle electrodes also showed, but to a lesser extent, significant levels of luciferase expression (p <0.05).

Similar results were obtained in immunogenicity studies in HLA-B7 mice. When mice were vaccinated intradermally with INVAC-1 followed by electrotransfer to the skin, the highest mean frequency of III + - specific CD8 T cells was detected using plate electrodes, and this difference was statistically significant compared to the PBS control group (p <0.05 ) (Fig.3B).

Thus, the plate electrodes showed the highest electrotransport capacity of pCMV-luc and the generation of significant levels of hTERT-specific CD8 T cells.

Localization of luciferase after ID injection, followed by gene electrotransfer

It is known that gene electrotransfer is very efficient in muscle (Andre et al., 2008). To ensure that after the ID injection of pCMV-luc, the luciferase gene was electrically transferred only to the skin, the skin flap was opened on the flank of C57BL / 6J mice at the treatment site, and the bioluminescence of the skin flap and underlying muscles was measured four days after gene electrotransfer. For this gene electrotransfer study, plate electrodes were used. The authors confirmed that the expression of the transgene occurred only in the skin and was not detected in the underlying muscles (Fig. 4).

HV pulse selection.

The first optimization of electrical parameters consisted in determining the most effective HV pulse amplitude (100 μs duration) from the following field amplitudes: 600, 800, 1000, 1200, 1400, 1600 V / cm. The intensity of the LV pulse (duration 400 ms) was kept constant at

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140 V / cm and the interval between HV and LV pulses was set to 1000 ms. This assessment was performed using the luciferase reporter gene and C57BL / 6J mice.

C57BL / 6J mice electrotransferred at 1200, 1400 and 1600 V / cm showed the most significant enhancement of luciferase expression compared to control mice (p <0.001) (FIG. 5). In particular, the highest average bioluminescence was obtained in the group treated at 1400 V / cm, and there was also a higher uniformity of results for this group compared to other groups. However, there was no statistically significant difference between the responses obtained from these three groups, i.e. 1200, 1400 and 1600 V / cm.

Selection of the best combination of HV-LV impulses.

The effect of HV-LV combinations was assessed by pCMV-luc electrotransfer and by INVAC-1-induced specific cellular immune responses after ID injection into the skin of C57BL / 6J mice. With regard to the HV pulse, 1000 or 1400 V / cm were chosen to be combined with different LV pulses. Immediately after the ID injection pCMV-luc or INVAC-1, one HV pulse (100 μs duration) at 1000 or 1400 V / cm was applied followed by one LV pulse (400 ms duration) at 60, 100, 140, 180 or 220 V / cm ... Ten combinations of HV-LV pulses were named from P1 to P10 (see table).

HV-LV pulse combinations evaluated in bioluminescence and ELISpot assays

(In / cm) LV = 60 LV = 100 LV = 140 LV = 180 LV = 220 HV = 1000 Pl P2 P3 P4 P5 HV = 1400 P6 P7 P8 P9 P10

Due to technical limitations, the Cliniporator® was unable to deliver consistently 220 V / cm for 400 ms. Thus, the results obtained using conditions P5 and P10 turned out to be unreliable and were excluded from the data analysis.

The three combinations of HV-LV pulses that generated the highest average bioluminescence intensities were P4, P8 and P9 (FIG. 6A). All of these three combinations showed high statistical differences compared to injection of pCMV-luc without EP (p <0.001). In particular, P9 showed the best average bioluminescence intensity, the highest minimum bioluminescence intensity, and the lowest dispersion point.

Then, the combinations of HV-LV pulses P4, P8 and P9 were tested against the INVAC1 vaccination ID. The intensity values of hTERT-specific CD8 T cell responses in these groups were comparable to the P3 combination previously described for DNA electrotransfer into subcutaneous tissues (Andre et al., 2008). Analysis of the data obtained as a result of the analysis of immunogenicity showed that the best combinations are P8 and P9, which ensured the generation of significant frequencies of N ^ + - specific CD8 T cells compared to control mice (p <0.01 and p <0.001, respectively) (Fig. 6B). Even though the difference between the P8 and P9 groups was not statistically significant, P9 showed a higher mean frequency of hTERT-specific CD8 T cells.

Taking into account the data of bioluminescence and immunogenicity analyzes, the best combination of HV-LV impulses was the P9 combination, i.e. one HV pulse (100 μs duration) at 1400 V / cm followed by an LV pulse (400 ms duration) at 180 V / cm.

Conclusion.

This study optimized the procedure for in vivo electrotransfer of the luciferase gene to the dermis and telomerase-based ID vaccination. Non-invasive plate electrodes delivering one high voltage pulse of 100 μs followed by one low voltage pulse of 400 msec displayed the highest levels of luciferase expression and the highest number of telomerase-specific CD8 T cells. The results obtained using this telomerase-based DNA vaccine could help develop a global DNA vaccination procedure using electrotransfer technology regardless of the antigen, whether it is a tumor antigen or a viral or bacterial antigen.

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Claims (15)

CLAIM 1. A method for preventing, suppressing or reducing a tumor in a subject in vivo, comprising the introduction of a nucleic acid encoding a tumor antigen and inducing a cellular immune response by intradermal injection and electrical permeabilization of skin cells using a single pulse of a high voltage electric field in the range from 1100 to 1400 V / cm and duration from 10 to 1000 μs; the subsequent single pulse of a low voltage electric field in the range from 100 to 200 V / cm and duration from 300 to 800 ms. 2. The method of claim 1, wherein the single low voltage pulse has an electric field strength of 180 V / cm. 3. A method according to any one of claims 1 or 2, wherein the single high voltage pulse has an electric field strength of 1250 V / cm. 4. A method according to any one of claims 1 to 3, wherein the single low voltage pulse has a duration of 350 to 600 ms. 5. The method of claim 4, wherein the single low voltage pulse has a duration of 400 ms. 6. A method according to any one of claims 1 to 5, wherein the single high voltage pulse has a duration of 50 to 150 μs. 7. The method of claim 6, wherein the single high voltage pulse has a duration of 100 μs. 8. A method according to any one of claims 1 to 7, wherein the electrodes to be used are non-invasive plate electrodes. 9. A method according to any one of claims 1 to 8, wherein the high voltage pulse and the low voltage pulse are separated from each other by a time interval ranging from 300 to 3000 ms. 10. The method of claim 9, wherein the time interval is 500 to 1200 ms. 11. The method of claim 10, wherein the time interval is 1000 ms. 12. A method according to any one of claims 1-11, wherein the nucleic acid encodes a TERT protein. 13. A method of antitumor vaccination or gene therapy of a subject in vivo, comprising the introduction of a nucleic acid encoding a tumor antigen and inducing a cellular immune response by intradermal injection and electrical permeabilization of skin cells using a single pulse of a high voltage electric field in the range from 1100 to 1400 V / cm and duration from 10 to 1000 μs; - 13 035485 of a subsequent single impulse of a low voltage electric field in the range from 100 to 200 V / cm and duration from 300 to 800 ms. 14. The method of claim 13, wherein the high voltage pulse and the low voltage pulse are separated by a time interval of 300 to 3000 ms. 15. A method according to any one of claims 1-14, wherein the cellular response is a CD8 T cell response.